In Trypanosoma brucei mitochondria, RNAs are synthesized polycistronically. Nevertheless, levels of mature monocistronic RNAs vary dramatically between life cycle stages. This indicates that steady-state RNA abundance, and thus gene expression, is controlled by posttranscriptional processes and these processes are developmentally regulated. Our hypothesis is that RNA decay pathways are critical determinants of gene regulation in this system. During the previous funding period, we identified distinct decay pathways for edited and unedited RNAs. Polyadenylation stimulates turnover of unedited RNAs while, conversely, dramatically inhibiting decay of edited RNAs, including those that are minimally edited. Our data suggest a model involving an exoribonuclease with intrinsic specificity for polyadenylated RNA, as well as sequence specific RNA binding proteins that prevent decay of their poly(A+) edited RNA targets. In organello data also implicate the RET1 terminal uridylyl transferase in facilitating turnover of unedited poly(A+) RNA. In our search for trans-acting factors involved in RNA turnover, we identified T. brucei homologs of the yeast mitochondrial degradosome components, DSS-1 exoribonuclease and SUV3 RNA helicase, and we showed that TbDSS-1 is essential for growth and involved in regulating mitochondrial RNA abundance. The long-term goal of this project is to understand the roles of RNA turnover in trypanosome mitochondrial gene expression, and to elucidate the underlying biochemical mechanisms that regulate these events. Aims proposed for the next funding period are: 1) Analyze the mechanism and machinery of unedited RNA turnover. We will identify poly(A)-selective exoribonucleases and characterize their function using RNAi-mediated targeted depletion. Analysis of RET1 RNAi cells will allow us to define the RNAs whose turnover is impacted by RET1 in vivo and assess 3' end modifications that may govern turnover rates. 2) Analyze the mechanism and machinery of edited RNA turnover. We will determine the range of edited RNAs that are stabilized by polyadenylation, and precisely define cis-acting stabilization sequences for a subset of these RNAs. Proteins that specifically bind the RPS12 stabilization determinant will be identified. 3) Determine the structure and function of the mitochondrial degradosome using a combined affinity chromatography and RNAi approach.